The ability to conduct non-invasive and comprehensive microscopy of entire internal organ systems in living human patients would have a profound impact on our ability to detect pre-malignant conditions and cancer at an early stage. Two complementary optical biopsy techniques, optical coherence tomography (OCT) and reflectance confocal microscopy (RCM), have shown promise for obtaining microscopic images in vivo. However, since OCT provides architecture and RCM cellular structure, neither method alone supplies all of the information that is required for accurate morphologic diagnosis. In addition, due to their limited fields of view, these techniques currently suffer from the same point-sampling limitations as conventional biopsy. In this proposal, we will merge new high-speed forms of these two technologies, spectrally encoded confocal microscopy (SECM) and spectral-domain OCT (SD-OCT), to create an endoscopic microscopy system that is capable of comprehensively imaging tissue architectural and cellular structure over extremely large tissue areas. Due to the considerable need for better methods to diagnose Barrett's esophagus (BE) and our expertise in esophageal imaging, the clinical focus of this grant will be the development of a device for improving screening for BE and surveillance of BE patients. The Aims of this proposal are motivated by our extensive prior experience investigating the diagnostic potential of endoscopic OCT and our recent work developing high-speed, large field-of-view SD-OCT. Although we have previously demonstrated the principles of SECM, the development of an endoscopic SECM probe and system capable of imaging large tissue volumes presents significant engineering challenges. The R21 phase of this work will focus on these challenges by developing a working system and probe that contain the key components required for wide-field endoscopic confocal microscopy. Also in the R21, we will evaluate the capability of SECM to visualize subcellular morphology relevant to BE, another important milestone in the critical path towards clinical viability. In the R33 phase, we will develop a hybrid imaging system and probe that complements the subcellular resolution of SECM with co-registered images of architectural structure obtained by SD-OCT. In the end, we will demonstrate comprehensive human esophageal imaging in vivo, covering the full range of spatial scale required for accurate morphologic diagnosis. While targeted to reduce the mortality of esophageal adenocarcinoma, this technology will also provide a platform for large-area microscopic screening in a variety of other organs, such as the colon, cervix, and bladder, where screening large tissue areas for early cancer is presently underserved by existing diagnostic paradigms. [unreadable] [unreadable] [unreadable]